The present invention relates to biological hydrogen production. More specifically, the present invention relates to a process for employing the components of photosynthetic organisms, to use light energy to make molecular hydrogen.
Aerobic oxygen-producing photosynthetic organisms, or subcellular components from such organisms, have been used previously to make hydrogen gas (Benemann et al., Proc. Nat. Acad. Sci. USA 70:2317, 1973, Rosen and Krasna, Photochem. Photobiol. 31:259, 1980, Rao et al, Biochimie 60:291, 1978).
The components of cell-free (i.e., in vitro) systems reported in these references require isolated thylakoids or solubilized photosystem I (PSI) from thylakoids; an electron donor, such as water or an artificial electron donor such as dithiothreitol or ascorbic acid; a hydrogenase capable of accepting electrons from photosystem I that can catalyze the combination of 2 electrons and 2 protons to form molecular hydrogen when electrons are received from an electron donor that can be oxidized by the hydrogenase; and an exogenous electron carrier that is capable of accepting electrons from photosystem I and can donate electrons to the hydrogenase. Examples of exogenous electron carriers that have been used as electron donors to the hydrogenase include ferredoxin and cytochrome c3 and the dye methyl viologen.
The biochemical pathway for molecular hydrogen production from elemental hydrogen is known. An electron on photosystem I (either isolated photosystem I or photosystem I in thylakoids) is excited, typically by light, to a higher energy (lower redox potential) resulting in the donation of an electron to an exogenous electron carrier that in turn can transfer electrons to the enzyme hydrogenase. In this process, oxidized photosystem I can then extract an electron from an electron donor, either directly or through an electron transfer chain, such as that found in the thylakoid membrane. Where water acts as the electron donor, the electron transfer chain includes photosystem II. Meanwhile, two reduced electron carrier molecules (having been reduced by illuminated photosystem I) are able to donate electrons to the enzyme hydrogenase. Hydrogenase combines two electrons with two protons to form a hydrogen molecule.
The yield of molecular hydrogen from this process is limited because the exogenous electron carriers donate their electrons to destinations other than hydrogenase. For example, reduced electron carriers, such as ferredoxin and methyl viologen, will react with oxygen to form superoxide and hydrogen peroxide. This results in lost reducing power and decreased molecular hydrogen yield. Even in the absence of oxygen, this system is still inefficient because exogenous electron carriers in this system oxidize to a large extent without reacting with hydrogenase (Rosen and Krasna, Photochem. Photobiol. 31:259, 1980). Exogenous electron carriers can be oxidized by oxidized photosystem I itself to produce what is known as cyclic electron transfer. This results in the oxidation of the exogenous electron carriers. Any other enzymes or chemicals present that are capable of oxidizing the exogenous electron carriers could also compete with hydrogenase for those electrons, decreasing the yield of molecular hydrogen.
It is believed that exogenous electron carrier molecules are necessary to catalyze the electron transfer from photosystem I to hydrogenase. Perhaps because of that belief, low concentrations of thylakoids (or photosystem I) and hydrogenase have been used for in vitro hydrogen gas production. For example, thylakoids containing fifty micrograms (xcexcg) of chlorophyll per ml or approximately 0.05 micromolar (xcexcM) photosystem I was used for light-dependent molecular hydrogen production in Rao et al. (supra), and 0.7 units of hydrogenase per ml were used by Benemann et al. (supra).
A small amount of molecular hydrogen production has been observed in vitro in the absence of an exogenous electron carrier (Rosen and Krasna, Photochem. Photobiol. 31:259, 1980). This work did not include the use of a purified hydrogenase; rather, crude extracts of Clostridium pasteurianum were used in place of a purified enzyme. These systems contain a large amount of ferredoxin, a known exogenous electron carrier and, indeed, the authors attributed hydrogen production to ferredoxin contamination. This explanation is further supported by the fact that the system of Rosen and Krasna (supra) would have contained much more ferredoxin than hydrogenase in the C. pasteurianum extracts.
Hoffman et al. (Z. Naturforsch 32c:257, 1977) have also reported a small amount of light-driven molecular hydrogen production from an in vitro system using thylakoids and hydrogenase from C. pasteurianum. Here, the rate of light-driven hydrogen production increased linearly with rising exogenous electron carrier concentration and could be extrapolated approximately to zero at zero added exogenous electron carrier (see FIG. 2, of that reference). Again, the small amount of hydrogen production observed in this system without an exogenous electron carrier could be explained, as above, by ferredoxin contamination of either thylakoids or hydrogenase.
At least two groups of oxygen-producing photosynthetic organisms are capable of producing hydrogen in vivo. These include cyanobacteria and green algae. Cyanobacteria generally use the enzyme nitrogenase to produce molecular hydrogen. Electrons used in this molecular hydrogen-producing process are derived from stored carbohydrate and are used to reduce ferredoxin, which is the immediate electron donor for nitrogenase (reviewed by Markov et al. Advances in Biochemical Engineering and Biotechnology 52:60, 1995). Hydrogenase can also catalyze molecular hydrogen production in cyanobacteria (supra). In most cases the electrons for molecular hydrogen production are obtained from stored carbohydrate. In cyanobacteria, molecular hydrogen production is inhibited by oxygen and/or light. Light-dependent hydrogenase-mediated molecular hydrogen production occurs in Oscillatoria limnetica in photosystem I-dependent reactions when the cells are in the presence of sulfide, which inactivates photosystem II and prevents oxygen production. The pathway of electron transfer from photosystem I to hydrogenase is unknown.
Green algae can also photoevolve (i.e., produce) molecular hydrogen via hydrogenase. The pathway of electron transfer is again unknown and in one case does not depend on photosystem I (Greenbaum et al. Nature 376:438, 1995). The source of electrons for the process has been shown to be endogenously fermented carbohydrate (Klein and Betz, Plant Physiol. 61:953, 1978). Hydrogen production stops in the presence of carbon dioxide (Vatsala and Seshadri, Proc. Indian Nat""l Sci. Acad. B51:282, 1985), indicating that the electron sink of carbon dioxide reduction is a better competitor for photosynthetic electron flow than hydrogenase.
Molecular hydrogen has a number of commercial uses. Molecular hydrogen is used for the production of ammonia, in petroleum refining, in the food industry for hydrogenation of vegetable oils, in electronic circuitry manufacture and as a fuel, for example, for space travel. Hydrogen is an almost pollution-free fuel and when burned, hydrogen produces only water vapor. Molecular hydrogen is currently made primarily by steam reforming of natural gas but this process produces carbon dioxide.
There is a need for new methods for producing molecular hydrogen since steam reforming of natural gas results in the production of carbon dioxide, natural gas is not a renewable resource, and carbon dioxide has been associated with global warming.
This invention relates to methods for producing molecular hydrogen and for obtaining hydrogen gas. The process uses a hydrogenase enzyme and components of the photosynthetic apparatus from oxygen-producing photosynthetic organisms. The methods and compositions of this invention do not require an exogenous electron carrier.
In one aspect of this invention a method for producing molecular hydrogen is disclosed. The method comprises the steps of combining (a) photosystem I from a photosynthetic organism, (b) at least one hydrogenase enzyme wherein the enzyme is capable of catalyzing the production of hydrogen gas, and (c) an electron donor capable of donating electrons to photosystem I; and producing molecular hydrogen wherein molecular hydrogen production does not depend on the presence of an exogenous electron carrier. In a preferred embodiment of this aspect of the invention the photosystem I of the combining step is present in thylakoids and the electron donor includes the natural photosynthetic electron transfer process present on the thylakoid membranes. In this embodiment, the electron donor is preferably water. In one embodiment the electron donor of the combining step is capable of donating electrons indirectly to photosystem I. In a preferred embodiment, the indirect electron donor is water. In another embodiment the electron donor of the combining step is capable of donating electrons directly to photosystem I. The direct electron donors include a variety of donors including dithiothreitol, ascorbic acid, and the like.
In one embodiment, molecular hydrogen production occurs in a cell-free composition. In another embodiment, the photosystem I of the combining step is an isolated photosystem I complex. The hydrogenase of the combining step can be purified from a cell or is an isolated recombinant protein.
In one embodiment, the electron donor of the combining step is dithiothreitol, in another the electron donor of the combining step is dithionite. The electron donor can be a combination of electron donors, including, but not limited to dithiothreitol and ascorbic acid.
In one embodiment of the method of this invention, the method includes, after the combining step, the step of exposing the composition to light. The producing step can additionally comprise the step of isolating molecular hydrogen as a gas.
This invention also relates to a cell free composition capable of producing molecular hydrogen comprising: photosystem I from a photosynthetic organism; at least one hydrogenase enzyme, wherein the enzyme is capable of catalyzing the production of hydrogen gas; and at least one electron donor capable of donating electrons to photosystem I wherein molecular hydrogen production is independent of an exogenous electron carrier. In one embodiment the photosystem I is present in isolated thylakoids in the composition and in another embodiment, the photosystem I is present in the composition as an isolated complex, preferably substantially thylakoid membrane free.
In one embodiment, the hydrogenase is purified from a cell and in another embodiment, the hydrogenase is a recombinant protein. In one embodiment the electron donor is water and the photosystem I complex is present in isolated thylakoids in the composition. In another embodiment the electron donor is dithiothreitol or dithionite. In yet another embodiment, the electron donor is a combination of dithiothreitol and ascorbic acid.
In one embodiment of this composition capable of producing molecular hydrogen, the combination is exposed to light and in another embodiment, for example, where the electron donor is dithionite, the composition capable of producing molecular hydrogen is maintained in the dark.